Gut microbiota or flora consists of microorganisms that live in the digestive tracts of animals. The 10 trillion or so microbial cells living in the gut, exceed the number of human cells by 10 to 1.
In the 1960s, electron microscopy revealed that Gram-negative cells release extracellular vesicles (EV) or outer membrane vesicles (OMV). Extracellular vesicles are spherical with a size of 20-200 nm and consist of phospholipid bilayers. Gram-negative bacterial extracellular vesicles have various outer membrane proteins as well as LPS (E. Y. Lee et al., Proteomics in gram-negative bacterial outer membrane vesicles. Mass. Spectrom. Rev. 2008; 27(6):535-555). There are reports on the presence of meningococcal vesicles in the blood of patients with fatal sepsis (E. Namork and P. Brandtzaeg, Fatal meningococcal sepsis with “blebbing” meningococcus. Lancet. 2002; 360(9347):1741), and the ex vivo secretion of an inflammatory mediator from meningococcal extracellular vesicles (M. R. Mirlashari et al., Outer membrane vesicles from Neisseria meningitidis: effects on cytokine production in human whole blood. Cytokine. 2001; 13(2):91-97; A. Bjerre et al., Complement activation induced by purified Neisseria meningitidis lipopolysaccharide (LPS), outer membrane vesicles, whole bacteria, and an LPS-free mutant. J. Infect. Dis. 2002; 185(2):220-228). However, nowhere have gut flora-derived extracellular vesicles been reported to cause local diseases characterized by mucosal inflammation such as in gastritis, peptic ulcer, stomach cancer, inflammatory enterocolitis, colorectal cancer, etc., and systemic inflammatory diseases such as sepsis, arteriosclerosis, diabetes, etc. in previous literature.
Recently, the prevalence rate of sepsis has increased around the world because of people's defense systems against bacterial infection being weakened with an increase in the aged population and the use of immunosuppressants and anti-cancer agents. Sepsis is a potentially deadly disease that is characterized by a systemic inflammatory state resulting as a complication from the local bacterial or fungal infection (M. M. Levi et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit. Care. Med. 2003; 31(4):1250-1256). In sepsis, the substance that is introduced into the blood after being secreted by pathogens upon local infection and which is secreted by pathogens introduced into blood vessels activates intravascular inflammatory cells to induce systemic inflammatory response syndrome and simultaneously activates endothelial cells to cause disseminated intravascular coagulation and thrombosis. In addition, the substances secreted from the pathogens are assigned to the major organs such as the lung to provoke inflammation and corresponding tissue injury, with a mortality rate of 30% (E. Lolis and R. Bucala, Therapeutic approaches to innate immunity: severe sepsis and septic shock. Nat. Rev. Drug. Discov. 2003; 2(8):635-645).
Sepsis is defined as systemic inflammatory response syndrome in response to a pathogenic process. However, in over half the cases of sepsis no specific pathogen is identified (R. S. Munford, Severe sepsis and septic shock: the role of gram-negative bacteremia. Annu. Rev. Pathol. 2006; 1:467-496). This indicates that the direct introduction of pathogens into the blood is not essential for the onset of sepsis, suggesting that pathogen-derived substances introduced into the blood might be a cause of sepsis. For example, on the basis of the finding that when introduced into the blood, lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, causes sepsis, research has widely been conducted to develop sepsis therapeutics (S. M. Opal, The host response to endotoxin, antilipopolysaccharide strategies, and the management of severe sepsis. Int. J. Med. Microbiol. 2007; 297(5):365-377). However, no therapeutics targeting LPS have been successfully developed thus far (J. Hellman, Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in gram-negative sepsis and causes inflammation and death in mice. J. Biol. Chem. 2002; 19; 277(16): 14274-14280).
It is very important to construct proper animal models mimicking human diseases in developing technologies for diagnosis, prophylaxis and therapy of human diseases. To establish sepsis animal models, the following three methods have been employed so far (J. A. Buras et al., Animal models of sepsis: setting the stage. Nat. Rev. Drug. Discov. 2005; 4(10):854-865): intraperitoneal injection of LPS; intraperitoneal injection of pathogens; and cecal ligation and puncture (CLP). However, these sepsis animal models suffer from the drawback of being low in reproducibility and great in error rate between operators and lacking the ability to properly express the phenotype of sepsis. Hence, an animal model that is highly reproducible and able to sufficiently express the phenotype of human sepsis, with a low error guaranteed between operators is required for the development of techniques for the diagnosis, prevention and treatment of sepsis.
An increase in blood inflammatory cytokine (particularly, IL-6) level is the hallmark of sepsis. There is known a method for evaluating the effect of therapeutic candidates on bacterial infection by measuring the level of the inflammatory factors induced by bacteria (PCT International Patent Publication No. WO2009/030093 Functions and uses of human protein phosphatase 1 inhibitor-2). However, little is known about any method for ex vivo screening candidate drugs capable of regulating inflammatory mediators by applying gut flora-derived extracellular vesicles to cells or for in vivo screening candidate drugs capable of regulating inflammatory mediators by applying the candidate drugs to a sepsis animal model established with gut flora-derived extracellular vesicles.
From decades ago, vaccines based on bacterial exotoxin proteins have been developed and used. Vaccines against Gram-positive bacteria have been developed on the basis of capsular polysaccharides, but suffer from the disadvantage of inducing the formation of antibodies independent of T cells. Developed to avoid this problem were vaccines in which a protein is conjugated with a capsular polysaccharide. These types of vaccines, however, work only on a sub-type of specific bacteria. So far, no clinical cases of using vaccines against Gram-negative bacteria have been reported. Recently, a vaccine against the Gram-negative bacterium meningococcus was developed from the artificial vesicles that were produced by treating the bacteria with a detergent (M. P. Girard et al., A review of vaccine research and development: meningococcal disease. Vaccine. 2006; 24(22):4692-4700). U.S. Pat. No. 7,384,645 “Outer membrane vesicles from Gram negative bacteria and use as a vaccine” discloses the use of the vesicles derived from meningococcus as a vaccine. U.S. Pat. Publication No. US 2007/0166333 “Method of antigen incorporation into neisseria bacterial outer membrane vesicles and resulting vaccine formulations” addresses a method for the incorporation of a protein antigen into meningococcal vesicles whereby the resulting vesicles maintain the immunogenicity and immunostimulatory properties of the vesicles and generate a superior immune response and thus can be used as a vaccine for the prophylaxis and therapy of meningococcal infection. In addition, with regard to the production and use of meningococcal vaccines, a process of making novel engineered meningococcal strain which is suitable for the production of meningococcal vaccines is disclosed (PCT International Patent Publication Nos. WO 2007/144316 and WO 2004/014417). Further, salmonella-derived vesicles were evaluated as a vaccine for activating congenital and acquired immune responses of hosts (R. C. Alaniz et al., Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo. J. Immunol. 2007; 179(11):7692-701). No reports have thus far been suggested on the use of extracellular vesicles as a vaccine for the prophylaxis and therapy of diseases caused by gut flora-derived extracellular vesicles and infection by gut flora.